Biology subjects

Schmidt, J.

Publications and source records attributed to Schmidt, J..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

The origin and molecular evolution of the mammalian liver cell architecture

The liver is a central organ with essential roles in processes such as nutritional metabolism, detoxification, and immune defense1-6. It has been instrumental in the adaptation of mammalian species to diverse environments, as reflected by its rapid molecular evolution7,8. However, the origins and evolutionary dynamics of liver cell types and their structural organization remain largely unexplored. Here we report evolutionary analyses of transcriptome and chromatin accessibility data for liver cells from 17 species, spanning all major feeding strategies (herbivory, omnivory, carnivory, insectivory), great apes (including humans), placental clades (Afrotheria, Xenarthra, Laurasiatheria, Euarchontoglires), major mammalian lineages (placentals, marsupials, monotremes), and a bird as outgroup. Integrated with spatial transcriptomics, our data reveal that liver zonation--the compartmentalization of hepatocyte functions along the lobule, the livers fundamental anatomical and functional unit--is conserved across mammals but absent in other vertebrates. We find that zonation originated in the mammalian ancestor, driven by the emergence of WNT and R-spondin signaling from central vein endothelial cells, which activate central hepatocyte gene expression via the transcription factor TCF7L2. Despite this conserved architecture and signaling, genes with zonated expression exhibit rapid evolutionary turnover. Consistently, hepatocytes evolve fast, likely due to reduced selective constraints, enabling adaptive changes under positive selection. Alongside immune cells, hepatocytes are therefore key drivers of the livers rapid evolution and functional innovations. In great apes, we identify human-specific shifts in zonation and cell-type-specific expression linked to recent cis-regulatory changes, particularly in genes involved in lipid metabolism, likely contributing to human-specific metabolic traits. Our study uncovers the origins of a mammal-specific liver cell architecture, within which reduced constraints facilitated molecular changes underlying ecological adaptations.

evolutionary biology↗

bioRxiv2025

Leveraging protein representations to explore uncharted fold spaces with generative models

A major challenge in computational de novo protein design is the exploration of uncharted areas within the protein structural space. However, the large degrees of freedom of protein backbones complicate the sampling process during protein design. Machine learning-based models have made great strides in this problem, however due to their nature they tend to exploit rather than explore the data distribution used for training the neural networks. To address some of these challenges, we propose a new coarse grained protein structure representation generative method, DiffTopo, a diffusion model which increases the sampling efficiency and diversity. Combined with a backbone level protein generative model like RFdiffusion, novel protein folds can be generated rapidly, allowing for efficient exploration of the designable topology space. Interestingly, we have discovered that by mirroring the topological organization of native proteins using a pipeline named MirrorTopo, we can readily expand the known fold space. We generated and experimentally characterized 30 different novel topologies from DiffTopo and 6 different novel mirror topologies from MirrorTopo. The developed framework relying on low resolution sampling provides new means for fold exploration challenges, which could in principle enhance our knowledge of the first principles of protein structure and folding, as well as create new opportunities for functional design.

bioinformatics↗

bioRxiv2025

Metabolic role of the LutACB operon in Neisseria gonorrhoeae resistance to neutrophil-mediated clearance

Neisseria gonorrhoeae, an obligate human pathogen, causes the sexually transmitted disease gonorrhoea and primarily colonizes the human genitourinary tract. An infection triggers a strong neutrophil-driven inflammatory response; however, viable gonococci are frequently recovered from neutrophil-rich patient exudates, indicating that these pathogens can survive neutrophil-mediated killing in vivo. To identify bacterial factors involved in this interaction, we screened a N. gonorrhoeae transposon library for mutants with reduced survival in polymorphonuclear leukocytes (PMNs). Among the identified candidates was NGFG_RS05085 (lutB), encoding an L-lactate oxidation FeS protein within the LutACB operon, which includes lutA (NGFG_RS05075), an FeS-binding protein, and lutC, the lactate utilisation protein C, (NGFG_RS05080). Deletion of lutA, lutB, or lutC did not significantly impair growth in standard culture medium or in minimal medium complemented with glucose, L-Lactate, or pyruvate. lutB and lutC, but not lutA, knockout significantly reduced bacterial survival within neutrophils compared to the wild-type strain. Polar effects of the mutations were ruled out by monitoring gene expression in deletion mutants. Metabolic profiling revealed disruption in nucleotide and glutathione synthesis, connecting the LutACB operon with sulfur metabolism, which could be required for a defence against neutrophil-derived reactive oxygen species (ROS). These findings highlight the important novel role of the LutACB operon, which connects lactate utilization and sulfur metabolism, and enables N. gonorrhoeae to withstand neutrophil-mediated oxidative stress during infection.

microbiology↗

bioRxiv2025

The evolution of gene regulatory programs controlling gonadal development in primates

Sex-determining pathways produce dimorphic gonads (ovaries and testes), yet the gene regulatory programs governing gonadogenesis and their evolution in primates remain little explored. Here we report evolutionary analyses of transcriptome and chromatin accessibility data of male and female human, marmoset (New World monkey), and mouse gonadal cells spanning key prenatal stages. We find that the two primates and mouse share similar X chromosome expression dynamics, including X chromosome reactivation (XCR), and that in Klinefelter syndrome (XXY) testes, germ cells undergo female-like XCR and escape of X inactivation. New male-specific regulatory regions have emerged progressively during mammalian evolution, especially on the X following sex chromosome origination. Further analyses revealed that male-specific regulatory regions evolved faster than female-specific ones in both supporting and pre-meiotic germ cells. However, female meiotic germ cells show even higher rates of molecular evolution and exhibit a permissive chromatin state that facilitates the birth of new genes, thus resembling their adult spermatogenic counterparts. Finally, we traced both conserved and species-specific gene expression trajectories across the three mammals, uncovering candidate genes for disorders of sex development that are typically central to cell-type-specific regulatory networks. Together, our study unveils both ancestral mammalian and recently evolved gene regulatory programs that control human- and primate-specific aspects of gonadal development in both sexes.

evolutionary biology↗

bioRxiv2025

Humanized Caffeine-Inducible Systems for Controlling Cellular Functions

Current cell therapies are limited by the lack of tools for controlling gene expression using humanized systems responsive to non-toxic stimuli. Starting from nanobodies that homodimerize in response to caffeine, we computationally designed inducible heterodimers and humanized the best-performing pairs. We used the resulting caffeine-inducible domains in engineered cytokine receptors for caffeine-inducible STAT3 signaling and in split transcription factors (caff-TFs) containing human-derived zinc-finger proteins. Heterodimerization of split transcription factors drastically enhanced their performance compared to homodimerization. We demonstrate that caff-TFs are compatible with lentiviral and retroviral delivery to Jurkat T-cells and enable inducible expression of therapeutic genes such as chimeric antigen receptors (CARs) in response to caffeine concentrations consistent with normal coffee consumption. By using the common, non-toxic molecule caffeine, and exclusively humanized protein components, these systems promise to be a safer alternative to existing systems and may be used in synthetic biology applications and for safer, more effective cell therapies.

synthetic biology↗

bioRxiv2025

The evolution of gene regulation in mammalian cerebellum development

Gene regulatory changes are considered major drivers of evolutionary innovations, including the cerebellums expansion during human evolution, yet they remain largely unexplored. In this study, we combined single-nucleus measurements of gene expression and chromatin accessibility from six mammals (human, bonobo, macaque, marmoset, mouse, and opossum) to uncover conserved and diverged regulatory networks in cerebellum development. We identified core regulators of cell identity and developed sequence-based models that revealed conserved regulatory codes. By predicting chromatin accessibility across 240 mammalian species, we reconstructed the evolutionary histories of human cis-regulatory elements, identifying sets associated with positive selection and gene expression changes, including the recent gain of THRB expression in cerebellar progenitor cells. Collectively, our work reveals the shared and mammalian lineage-specific regulatory programs governing cerebellum development.

evolutionary biology↗

bioRxiv2025

Finding space for rewilding: participatory scenarios reveal ecological opportunities based on plural values of nature

O_LIRewilding aims to restore degraded ecosystems in human-dominated areas by promoting self-sustained ecosystems and reducing human control over ecological processes. However, co-benefits and trade-offs between rewilding actions and the interests of local societies must be thoroughly considered. IPBES proposed the Nature Futures framework, a scenario tool to engage society in conservation planning. However, its potential for co-designing ecological restoration remains to be explored. C_LIO_LIThis paper presents a novel methodology that utilises the Nature Futures Framework to address opportunities, co-benefits and trade-offs using rewilding. The study aims to investigate the Nature futures Framework effectiveness in rewilding by involving stakeholders in participatory scenario development. To accomplish this, we conducted stakeholder interviews to co-design three distinct scenarios that reflect diverse values of nature and rewilding strategies for the German Oder Delta. C_LIO_LIThe scenario development process identified spatial priorities for three scenarios: Nature for Nature, Nature for Society (NfS) and Nature as Culture. (NaC). The first, focused on rewilding and increasing forest and river connectivity across 65.8% of the study area. NfS narratives covering 14.4% of the area emphasised de-intensification of forestry and reflooding of peatlands, while NaC prioritised transformation towards extensive agriculture and reflooding for paludiculture across 8.6%. C_LIO_LIThe study identified regions with single and multiple values of nature. Further, there were regions of overlap between scenarios, highlighting the interconnectedness of different rewilding actions and priorities. By pinpointing corresponding values, the research highlighted actions that could result in co-benefits. The findings emphasise the importance of considering pluralistic values when designing rewilding actions, which encourages stakeholders to highlight areas of less resistance to rewilding and co-adapt to the changes in the landscape. C_LIO_LIThis study presents a methodological framework for participatory scenario development for rewilding initiatives. By combining participatory processes with the Nature Futures Framework, our structured approach helps identify priority areas and navigate socio-ecological challenges in landscape restoration. It employs participatory mapping to aid the visualization of land-use changes and aligning rewilding actions with ecological and societal needs. This approach enhances governance frameworks and promotes inclusive rewilding efforts, creating a replicable model for landscape restoration that connects rewilding with sustainability and societal well-being goals. C_LI

ecology↗

bioRxiv2025

Cryptochrome 4b protein is likely irrelevant for the radical pair based magnetoreception in the European robin

Avian cryptochrome 4 (Cry4) protein is a putative magnetosensitive molecule facilitating precise long-distance navigation in migratory birds. Two splice variants of Cry4 were reported in European robin (Erithacus rubecula), namely ErCry4a and ErCry4b. It is known that ErCry4a protein exhibits electron transfer between the flavin adenine dinucleotide (FAD) cofactor and tryptophan residues that generates magnetically sensitive radical pairs for magnetoreception. However, little is known about the ErCry4b isoform. We therefore characterized the properties of ErCry4b to see whether it fulfills prerequisites to be a radical pair magnetic sensor molecule. Our results show that ErCry4b protein does not bind FAD in vitro. Computational structure simulations revealed that the FAD non-binding in ErCry4b is likely due to protein structure dynamics. Furthermore, ErCry4b protein abundance in the robin retina, cerebellum and liver is below the detection limit of immunoprecipitation assays coupled with mass spectrometry. Meanwhile, transcript analyses show that ErCRY4b mRNA abundance is 10 times less than ErCRY4b in the retina. In conclusion, ErCry4b does not fulfill the prerequisites to be a radical pair based magnetic sensing molecule due to the lack of FAD binding, and it might not even be expressed as a functional protein in the European robin.

biochemistry↗

bioRxiv2024

Structure-based Design of Chimeric Influenza Hemagglutinins to Elicit Cross-group Immunity

Antigenic variability among influenza virus strains poses a significant challenge to developing broadly protective, long-lasting vaccines. Current annual vaccines target specific strains, requiring accurate prediction for effective neutralization. Despite sequence diversity across phylogenetic groups, the hemagglutinin (HA) head domains structure remains highly conserved. Utilizing this conservation, we designed cross-group chimeric HAs that combine antigenic surfaces from distant strains. By structure-guided transplantation of receptor-binding site (RBS) residues, we displayed an H3 RBS on an H1 HA scaffold. These chimeric immunogens elicit cross-group polyclonal responses capable of neutralizing both base and distal strains. Additionally, the chimeras integrate heterotrimeric immunogens, enhancing modular vaccine design. This approach enables the inclusion of diverse strain segments to generate broad polyclonal responses. In the future, such modular immunogens may serve as tools for evaluating immunodominance and refining immunization strategies, offering potential to bridge and enhance immune responses in individuals with pre-existing immunity. This strategy holds promise for advancing universal influenza vaccine development. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/628867v2_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@18dec5borg.highwire.dtl.DTLVardef@98587forg.highwire.dtl.DTLVardef@1d9fb2dorg.highwire.dtl.DTLVardef@1f97884_HPS_FORMAT_FIGEXP M_FIG Graphical abstract: Overview of cross-group RBS transplantation approachPhylogenetically diverse HA strains can be incorporated into chimeric immunogens by RBS transplantation. The chimera are evaluated for cross-reactivity to subtype-specific antibodies and the ability to elicit neutralizing antibodies to multiple strains. C_FIG

biochemistry↗

bioRxiv2024

Mapping targetable sites on the human surfaceome for the design of novel binders

The human cell surfaceome, integral to cell communication and disease mechanisms, presents a prime target for therapeutic intervention. De novo protein binder design against these cell surface proteins offers a promising yet underexplored strategy for drug development. However, the vast search space and limited data on natural or competitive binders have historically limited experimental success. In this study, we systematically analyzed the entire human surfaceome, identifying approximately 4,500 targetable sites and introducing high-quality binder seeds tailored for protein design applications. To validate these seeds, we implemented two experimental approaches (protein scaffolding and peptide cyclization) on three representative targets (FGFR2, IFNAR2, and HER3). Our results revealed a high success rate, emphasizing the precision and therapeutic potential of these seeds, as well as the need for constant improvements of computational protein design pipelines utilizing machine learning and physics-based methods. Additionally, we present SURFACE-Bind, an interactive database offering open access to all generated data. The high-throughput computational design methods and target-specific binder seeds established here pave the way for a new generation of targeted therapeutics for the human surfaceome.

synthetic biology↗

bioRxiv2024

Convergence of autism proteins at the cilium

Hundreds of high-confidence autism genes have been identified, yet the relevant etiological mechanisms remain unclear. Gene ontology analyses have repeatedly identified enrichment of proteins with annotated functions in gene expression regulation and neuronal communication. However, proteins are often pleiotropic and these annotations are inherently incomplete. Our recent autism functional genetics work has suggested that these genes may share a common mechanism at the cilium, a membrane-bound organelle critical for neurogenesis, brain patterning, and neuronal activity-all processes strongly implicated in autism. Moreover, autism commonly co-occurs with conditions that are known to involve ciliary-related pathologies, including congenital heart disease, hydrocephalus, and blindness. However, the role of autism genes at the cilium has not been systematically investigated. Here we demonstrate that autism proteins spanning disparate functional annotations converge in expression, localization, and function at cilia, and that patients with pathogenic variants in these genes have cilia-related co-occurring conditions and biomarkers of disrupted ciliary function. This degree of convergence among genes spanning diverse functional annotations strongly suggests that cilia are relevant to autism, as well as to commonly co-occurring conditions, and that this organelle should be explored further for therapeutic potential. One-Sentence SummaryAutism genes of diverse functional annotations converge at cilia, deepening our understanding of underlying autism biology and co-occurring conditions.

cell biology↗

bioRxiv2024

The Spc105-Kre28 complex promotes mitotic error correction by outer kinetochore recruitment of the Ipl1/Aurora B kinase

Kinetochores link chromosomes to dynamic microtubules of the mitotic spindle. To ensure equal chromosome segregation, sister chromatids must achieve biorientation. The conserved kinase Aurora B phosphorylates outer kinetochore proteins on attachments lacking tension, allowing re-establishment of new connections until biorientation is achieved. Aurora B localizes to the centromere as part of the chromosomal passenger complex (CPC), but the underlying recruitment pathways can be eliminated without disrupting biorientation. It therefore remains unclear how the kinase operates during error correction. Here, we identify the conserved Spc105/Kre28 complex as an outer kinetochore receptor of the Aurora kinase Ipl1 and its activator Sli15 in Saccharomyces cerevisiae. We show that mutations in the helix bundle domain of Spc105/Kre28 impair mitotic error correction, resembling the effects of ipl1 or sli15 mutants. The defects can be suppressed by artificial recruitment of Ipl1. In biochemical experiments Ipl1/Sli15 directly associates with Spc105/Kre28, and a conserved segment in the Sli15 central domain is crucially involved in the binding mechanism. These results have important implications for the mechanism of tension-dependent error correction during chromosome biorientation.

cell biology↗

bioRxiv2024

BindCraft: one-shot design of functional protein binders

Protein-protein interactions (PPIs) are at the core of all key biological processes. However, the complexity of the structural features that determine PPIs makes their design challenging. We present BindCraft, an open-source and automated pipeline for de novo protein binder design with experimental success rates of 10-100%. BindCraft leverages the weights of AlphaFold21 to generate binders with nanomolar affinity without the need for high-throughput screening or experimental optimization, even in the absence of known binding sites. We successfully designed binders against a diverse set of challenging targets, including cell-surface receptors, common allergens, de novo designed proteins, and multi-domain nucleases, such as CRISPR-Cas9. We showcase the functional and therapeutic potential of designed binders by reducing IgE binding to birch allergen in patient-derived samples, modulating Cas9 gene editing activity, and reducing the cytotoxicity of a foodborne bacterial enterotoxin. Lastly, we utilize cell surface receptor-specific binders to redirect AAV capsids for targeted gene delivery. This work represents a significant advancement towards a "one design-one binder" approach in computational design, with immense potential in therapeutics, diagnostics, and biotechnology.

bioinformatics↗

bioRxiv2024

Structure of the complete extracellular bacterial flagellum reveals mechanism for flagellin incorporation

The bacterial flagellum is essential for motility, adhesion, and colonization in pathogens like Salmonella enterica and Campylobacter jejuni. Its extracellular structure comprises the hook, hook-filament junction, filament, and filament cap. The native structures of the hook-filament junction and the cap remain elusive, leaving the molecular details of cap-mediated filament assembly largely uncharacterized. Here, we report the structure of the complete extracellular flagellum, encompassing the hook, hook-filament junction, filament, and cap. This structure reveals intermediates of filament assembly, providing a molecular blueprint for flagellin folding and insertion at the filament tip. Mutagenesis and functional assays demonstrate the crucial roles of the caps terminal regions in flagellin incorporation, and of the structural integrity of the hook-filament junction. Finally, the structure of the cap and hook-filament junction prior to filament assembly reveals the structural basis for the initiation of filament assembly. Collectively, this study provides comprehensive insights into flagellum assembly and how flagellin incorporation is coupled with its secretion.

microbiology↗

bioRxiv2024

Comparison of retinol binding protein 1 with cone specific G-protein as putative effector molecules in cryptochrome signalling

Vision and magnetoreception in navigating songbirds are strongly connected as recent findings link a light dependent radical-pair mechanism in cryptochrome proteins to signalling pathways in cone photoreceptor cells. A previous yeast-two-hybrid screening approach identified six putative candidate proteins showing binding to cryptochrome type 4a. So far, only the interaction of the cone specific G-protein transducin -subunit was investigated in more detail. In the present study, we compare the binding features of the G-protein -subunit with those of another candidate from the yeast-two-hybrid screen, cellular retinol binding protein. Purified recombinant European robin retinol binding protein bound retinol with high affinity, displaying an EC50 of less than 5 nM, thereby demonstrating its functional state. We applied surface plasmon resonance and a Forster resonance transfer analysis to test for interactions between retinol binding protein and cryptochrome 4a. In the absence of retinol, we observed no robust binding events, which contrasts the strong interaction we observed between cryptochrome 4a and the G-protein -subunit. We conclude that retinol binding protein is unlikely to be involved in the primary magnetosensory signalling cascade.

biochemistry↗

bioRxiv2024

European Robin Cryptochrome-4a Associates with Lipid Bilayers in an Ordered Manner, Fulfilling a Molecular-Level Condition for Magnetoreception

Since the middle of the 20th century, long-distance avian migration has been known to rely partly on the geomagnetic field. However, the underlying sensory mechanism is still not fully understood. Cryptochrome 4a (ErCry4a), found in European Robin (Erithacus rubecula), a night-migratory songbird has been suggested to be a magnetic sensory molecule. It is sensitive to external magnetic fields via the so-called radical-pair mechanism. ErCry4a is primarily located in the outer segments of the double cone photoreceptor cells in the eye, which contain stacked and highly ordered membranes that could facilitate the anisotropic attachment of ErCry4a needed for magnetic compass sensing. Here, we investigate possible interactions of ErCry4a with a model membrane that mimics the lipid composition of outer segments of vertebrate photoreceptor cells by using experimental and computational approaches. Experimental results show that the attachment of ErCry4a to the membrane could be controlled by the physical state of lipid molecules (average area per lipid) in the outer leaflet of the lipid bilayer. Furthermore, polarization modulation infrared reflection absorption spectroscopy allowed us to determine the conformation, motional freedom, and average orientation of the - helices in ErCry4a in a membrane-associated state. Atomistic molecular dynamics studies supported the experimental results. A [~]1000 kcal mol-1 decrease in the interaction energy as a result of ErCry4a membrane binding was determined compared to cases where no protein binding to the membrane occurred. At the molecular level, the binding seems to involve negatively charged carboxylate groups of the phosphoserine lipids and the C-terminal residues of ErCry4a. Our study reveals a potential direct interaction of ErCry4a with the lipid membrane and discusses how this binding could be an essential step for ErCry4a to propagate a magnetic signal further and thus fulfill a role as a magnetoreceptor.

biophysics↗

bioRxiv2024

Phage display profiling of CDR3β loops enables machine learning predictions of NY-ESO-1 specific TCRs

T cells targeting epitopes in infectious diseases or cancer play a central role in spontaneous and therapy-induced immune responses. T-cell epitope recognition is mediated by the binding of the T-Cell Receptor (TCR) and TCRs recognizing clinically relevant epitopes are promising for T-cell based therapies. Starting from one of the few known TCRs targeting the cancer-testis antigen NY-ESO-1157-165 epitope, we built large phage display libraries of TCRs with randomized Complementary Determining Region 3 of the {beta} chain. The TCR libraries were panned against the NY-ESO-1 epitope, which enabled us to collect thousands of epitope-specific TCR sequences. We then trained a machine learning TCR-epitope interaction predictor with this data and could identify several epitope-specific TCRs directly from TCR repertoires. Cellular binding and functional assays revealed that the predicted TCRs displayed activity towards the NY-ESO-1 epitope and no detectable cross-reactivity with self-peptides. Overall, our work demonstrates how display technologies combined with machine learning models of TCR-epitope recognition can effectively leverage large TCR repertoires for TCR discovery.

bioinformatics↗

bioRxiv2024

High-resolution respirometry reveals altered mammalian tissue ketone body oxidation in different cardiometabolic diseases

Background and aimsReduced mitochondrial function has been implicated in metabolic disorders like type 2 diabetes (T2D), obesity, and metabolic dysfunction-associated steatotic liver disease (MASLD), which are tightly linked to insulin resistance and impaired metabolic flexibility. However, the contribution of the ketone bodies (KBs) {beta}-hydroxybutyrate (HBA) and acetoacetate (ACA) as substrates for mitochondrial oxidative phosphorylation (OXPHOS) in these insulin resistant states remains unclear. MethodsTargeted high-resolution respirometry protocols were applied to detect the differential contribution of HBA and ACA to OXPHOS capacity in heart, skeletal muscle, kidney, and liver of distinct human and mouse cohorts with T2D, obesity, and MASLD. ResultsIn humans with T2D, KB-driven mitochondrial OXPHOS capacity was [~]30% lower in the heart (p<0.05) and skeletal muscle (p<0.05) compared to non-diabetic controls. The relative contribution of KB to maximal OXPHOS capacity in T2D was also lower in both the heart ([~]25%, p<0.05) and skeletal muscle ([~]50%, p<0.05). Similarly, in kidney cortex from high-fat diet-induced obese mice, both the absolute and relative contribution of KB to OXPHOS capacity was [~]15% lower (p<0.05). Finally, hepatic HBA-driven mitochondrial OXPHOS capacity was 29% lower (p<0.05) in obese humans with MASLD compared to humans without MASLD. ConclusionsMitochondrial KB-driven OXPHOS capacity is impaired in insulin resistant states in various organs in absolute and relative terms, likely reflecting impaired mitochondrial metabolic flexibility. Our data suggest that KB respirometry can provide a sensitive readout of impaired mitochondrial function in diabetes, obesity, and MASLD.

cell biology↗